Method for producing a gradient elution

ABSTRACT

The method of proportioning two liquids comprising periodically controlling the operation of a valving .[.means.]. .Iadd.arrangement .Iaddend.to control the amount of each liquid supplied to a mixing region during each period of valve operation to produce a supply of eluent having a precisely controlled time varying concentration of each liquid.

This is a division of application Ser. No. 36,633, filed May 12, 1970now U.S. Pat. No. 3,712,513.

BACKGROUND OF THE INVENTION

This invention relates to the field of elution. More specifically itrelates to a method and system for producing an eluent having a knownconcentration of different liquids. More specifically still, it relatesto a method and system for producing an eluent having a time varyingconcentration of different liquids.

The simplest elution system, used for example in a chromatographiccolumn, supplies an eluent comprising a single liquid to the column. Inmany instances, however, this single liquid is not effective in removingall of the desired material from the column, and a second liquid mustsubsequently be supplied. Alternately, an eluent having a setconcentration of the two liquids is used. More than two liquids can andhave been used, but for convenience in describing the prior art and ourinvention, we will limit the discussion that follows to a system usingtwo liquids which we will label liquid A and liquid B. Switching liquidsin mid-operation and the use of a mixture of liquids have not beenfavored procedurally, and there has been a recent tendency to gradientelution systems. In gradient elution, the initial eluent contains a setconcentration of the liquids (usually 100 percent A and none of B), andthis concentration is slowly altered, through intermediateconcentrations, to a second set concentration of the liquids (usuallynone of A and 100 percent of B). The conventional way of doing this isto use two constant flow pumps. One of these pumps pumps liquid from apot containing liquid having the initial concentration of the twoliquids (usually 100 percent of A and none of B) to the column. Thesecond pump pumps liquid having the desired final concentration of thetwo liquids (usually none of A and 100 percent of B) into the potprovided for the first pump so that the concentration of liquid in thepot which is being pumped to the column, gradually changed to the finalconcentration. This procedure has several disadvantages. Two pumps arenecessary, and although the change in concentration can be fairlyprecisely controlled, the system is not versatile in terms of theability to tailor changes in concentration, to meet the needs ofindividual tests. In some instances, instead of using the two stagesystem described above, two proportional flow valves are used incombination with the two pumps, and the flow of liquid through thevalves is varied to achieve the desired concentration. This systemsuffers from the fact that the flow through proportional valves isdifficult if not impossible to control with any precision, especially atlow flow, so that the concentration cannot be controlled precisely.

It is an object of the present invention to provide a method and systemfor providing an eluent in which the concentrations of the liquid partsis precisely known. It is a further object of the present invention toprovide an eluent having precisely controlled time varyingconcentrations of the liquid parts. It is a still further object of thepresent invention to provide a system for producing an eluent havingtime varying concentrations of liquid parts in which the means forcontrolling the concentration of liquids provides for flexible controlover the concentrations of the liquids in the eluent and over the rateof change of those concentrations. It is another object of the presentinvention to provide a method and system for producing an eluent havinga time varying concentration of liquids, in which a single pump is used.

SUMMARY OF THE DISCLOSURE

These objects are accomplished by providing separate sources of liquids,in the case of being discussed, a source of first liquid and a source ofsecond liquid; a proportioning means comprising a mixing region, valvingmeans connected to the mixing region and programming means connected toand adapted to activate the valving means; and separate liquid supplymeans, in this case a first liquid supply means and a second liquidsupply means to supply the first and second liquids respectively to thevalving means. This mixing region is then connected directly to thecolumn. The valving means comprises at least two operative portions, afirst operative portion connected between the first liquid supply meansand the mixing region and a second operative portion connected betweenat least a portion of the second liquid supply means and the mixingregion. The programming means periodically activates one or the other ofthe operative portions of the valving means to allow the mixing regionto be periodically supplied with each liquid.

The mixing region can be any region adapted to produce the requiredamount of mixing between the two fluids. In one embodiment the mixingregion can be a separate mixing chamber with a volume substantiallylarger than the maximum volume of liquid entering the mixing regionduring the period when either operative portion of the valving means isactivated, and the chamber can be provided with some means to thoroughlymix the liquids in the chamber so that the mixture is homogeneousthroughout the chamber. In a simpler but still effective embodiment, themixing region is provided by the connecting lines between the valvingmeans and the column alone. Depending upon how homogeneous the mixturemust be for the particular purpose involved, the volume of the mixingregion can be less than, equal to or greater than the maximum volumeentering the mixing region during the period when either operativeportion of the valving means is operative. While it might be expectedthat either a very large mixing region or some additional mixing device,such as frittered plug to provide highly turbulent regions would have tobe provided, we have found that except in those applications where ahigh degree of uniformity is required, a reasonably small volume ofconnecting tubing alone will provide substantial mixing. As an example,when each operative portion of the valving means passes 0.25 cc. ofliquid, a mixing region having a volume of 0.50 cc. will produce a finalmixture at the end of the mixing region having a periodic variation ofno more than 1.0 percent. If a variation of 10 to 20 percent weretolerable, the volume of the mixing region could even be less than thevolume of liquid periodically passed through the valving means. Itappears that in this dynamic system, substantial mixing is produced bydiffusion, friction, eddy currents and the like.

The mixing region is also provided with a means to allow a portion ofany liquid contained in the mixing region to be removed from the mixingregion, thereby supplying an eluent having a concentration of eachliquid equivalent to the instantaneous concentration of each liquid inthe mixing region at the time the liquid is removed from the mixingregion. Preferably the liquid is removed from the mixing region andsupplied to the column continuously.

The valving means can be: a three-way valve in which case one of thepaths therein is the first operative position and the other path is thesecond operative position; a pair of separate valves, in which eachvalve is a separate operative portion of the valving means, or anysuitable on-off valving means which will allow liquid to be switchedfrom one fluid line to another fluid line. For convenience in thediscussion that follows, we will limit our discussion to a system inwhich two separate valves are used. These valves can be any valve, suchas a solenoid valve or an air actuated valve which can be activated byan externally generated signal. The programming means is, then, anymeans that will generate and supply a signal to the valves which willactivate the valves. In the preferred embodiment the valves are solenoidvalves; and the programming means is an electronic signal generatoradapted to produce two electronic signals, one being a periodic signaland the other being a monotonically increasing function. The programmingmeans is further adapted to control the valves by opening one when theinstantaneous value of one signal exceeds the instantaneous value of theother signal, and then opening the other when the reverse situationoccurs.

The first and second liquid supply means can be two separate pumps,either constant flow or constant pressure pumps as the situationdemands; but in the preferred embodiment, a single pump is used. Insteadof the second pump, a holding chamber is provided. The single pump aloneis used as the first liquid supply means to supply the first liquid tothe first valve. The second liquid supply means comprises both theholding chamber, which is adapted to be filled with the second liquid,and the pump. The pump is connected to the holding chamber and thesecond valve in a manner such that when the second valve is open, thefirst liquid supplied by the pump is used to force the second liquidcontained in the holding chamber to the second valve. The valve can belocated on either the upstream or downstream side of the holdingchamber, and either a constant flow or constant pressure pump can beused depending on the particular needs of the system. The holdingchamber must be constructed so that substantial mixing of the fixedliquid with the second liquid does not occur in the true interval whilethe system is in operation. A holding chamber in the form of a long thintube which hold substantially more liquid than is to be used in aparticular situation fills this criteria.

The detailed operation of the system described above can best bedescribed by reference to the following figures:

FIG. 1 is a schematic diagram of the simplest embodiment of the presentinvention;

FIG. 2 is a schematic diagram of a first embodiment of an improvedversion of the present invention which requires the use of only onepump;

FIG. 3 is a schematic diagram of a second embodiment of the presentinvention which requires the use of only one pump;

FIG. 4 is a schematic diagram of the preferred embodiment of the presentinvention;

FIG. 5 is a schematic diagram of one embodiment of a device used tomeasure the amount of liquid removed from the holding chamber;

FIG. 6 is a diagram of several of the possible wave functions that canbe used to control the valves, and the programming sequence theyproduce; and

FIG. 7 is a graph of one possible programming sequence.

DETAILED DESCRIPTION OF THE DRAWINGS

Referring to FIG. 1, a source of liquid A, 11, and a source of liquid B,12, ar provided and connected respectively to constant pressure pumps13, and 14. These pumps are each connected to proportioning means 15which comprises two on-off valves 16 and 17, a mixing region, and aprogramming means (not shown) which is connected to the valves andadapted to control the operation of the valves. For convenience, themixing region is illustrated in this and the other figures as a separatemixing chamber 18 though, as discussed above, this is not necessary.Pumps 13 and 14 are connected respectively to valves 16 and 17 which areeach connected to mixing chamber 18. The mixing chamber is in turnconnected to a chromatographic column system 19 comprising an injectionport 20, a column 21, and a detector 22. The column system in turn isconnected to or empties into a drain 23. The elution system describedherein is useful in any application where a well controlled or changingconcentration of liquids in a mixture is required, but for convenience,we are discussing the system in its application to a system forsupplying the eluent used in liquid chromatography. Being included formerely descriptive purposes, the details of the chromotographic systemwill not be described.

The on-off valves 16 and 17 are normally closed on-off valves of a typecontrolled by an external signal. Solenoid valves or air actuated valvesare typical examples of such valves. Alternately, a signal actuatedthree-way valve could be used, or any valving means adapted to switchpositively from one liquid to the other at controlled intervals. Forconvenience, we will limit the discussion that follows to solenoidvalves and electronic programming means. It is to be understood,however, that any externally activated valve and a programming meansadapted to activate it can be used instead.

In operation, the constant pressure pumps supply their liquids atconstant pressure to each of the normally closed on-off valvesrespectively. The programming means then opens one of the valves for aset period of time. We will assume in what follows that liquid A isalways the first fluid to flow and consequently, that valve 16 is theone opened, but the reverse can be true. A set volume of liquid A willenter the mixing chamber. Then the programming means will deactivatevalve 16, and activate valve 17 for a second period of time to allow aset volume of fluid B to enter the mixing chamber. In the normalsituation, the initial mixture of liquids in the mixing chamber would be100 percent of liquid A and none of liquid B. To fill the chamberinitially, then, valve 16 would open either periodically or for a longduration until the mixing chamber was filled with liquid A, and valve 17would remain closed. The volume of the mixing chamber in this embodimentis larger than the volume of liquid each valve is programmed to allow topass during each of its cycles when the valves are in periodicoperation. During the filling step, instead of periodically opening andclosing valve 16 to fill the mixing chamber 18, valve 16 can be keptopen until the chamber is filled. In those situations where a highdegree of uniformity is required, only a fraction of the volume ofliquid needed to fill the mixing chamber is allowed to pass through thevalve. The eluent supplied to a chromatographic column can be suppliedat high pressure or just allowed to flow through the column under theforce of gravity. We will limit our discussion to the former, moredifficult situation, when the mixing chamber must be completely filledwith the initial concentration. In this situation, a bleed-off valve onthe mixing chamber may be useful to bleed off air trapped in the chamberor to assist in changing the fluids in the chamber. Assuming now thatthe mixing chamber is completely filled with liquid A, the programmingmeans operates to change the concentration in the mixing chamber whilethe liquid in the chamber is being supplied under pressure, the pressureof pumps 13 and 14, to the column. The simplest situation would be wherethe final mixture is to contain 100 percent of liquid B and none ofliquid A. One way to achieve this concentration would be to have theprogramming means activate valve 17 and not activate valve 16 at all. Ifsubstantial homogeneity of the mixture at the end of the mixing chambernext to the column is not achieved, then the precision andreproducibility of the concentration supplied to the column is poor andthe resulting chromatographic determinations suffer.

In the operation just discussed, valve 17 is periodically orcontinuously opened introducing small volumes of liquid B into theliquid in the mixing chamber. Gradually the concentration of liquid B inthe mixing chamber increases until the mixing chamber finally containsonly liquid B. It is apparent that in some cases the supply of liquid Acontained in the mixing chamber will not be sufficient to allow thechange from the initial concentration to the final concentration takesplace in a manner which will suit the needs of most eluting devices. Inthis case the programming means will alternately activate valve 16 andvalve 17 to maintain the pressure or just allowed to flow through thecolumn under the force of gravity. We will limit our discussion to theformer, more difficult situation, when the mixing chamber must becompletely filled with the initial concentration. In this situation, ableed-off valve on the mixing chamber may be useful to bleed off airtrapped in the chamber or to assist in changing the fluids in thechamber. Assuming now that the mixing chamber is completely filled withliquid A, the programming means operates to change the concentration inthe mixing chamber while the liquid in the chamber is being suppliedunder pressure, the pressure of pumps 13 and 14, to the column. Thesimplest situation would be where the final mixture is to contain 100percent of liquid B and none of liquid A. One way to achieve thisconcentration would be to have the programming means activate valve 17and not activate valve 16 at all. If substantial homogeneity of themixture at the end of the mixing chamber next to the column is notachieved, then the precision and reproducibility of the concentrationsupplied to the column is poor and the resulting chromatographicdeterminations suffer.

In the operation just discussed, valve 17 is periodically orcontinuously opened introducing small volumes of liquid B into theliquid in the mixing chamber. Gradually the concentration of liquid B inthe mixing chamber increases until the mixing chamber finally containsonly liquid B. It is apparent that in some cases the supply of liquid Acontained in the mixing chamber will not be sufficient to allow thechange from the initial concentration to the final concentration takesplace in a manner which will suit the needs of most eluting devices. Inthis case the programming means will alternately activate valve 16 andvalve 17 to maintain the supply of liquid A in the mixing chamber. Ifvalves 16 and 17 are alternately opened for the same set period of time,the final concentration will be 50 percent of liquid A and 50 percent ofliquid B. To achieve a final concentration of 100 percent liquid B, theperiod during which valve 16 is open must be gradually decreased tozero. This is usually accompanied by an increase in the period duringwhich valve 17 is open. The relative periods involved can be determinedby the needs of the system. In fact the periods need not vary. If a setconcentration is desired, each valve can be programmed to open for adifferent set period of time which will fix the desired concentration,and the concentration can be maintained by leaving the valve periodsfixed during the entire operation.

FIG. 7 illustrates the versatility of such a device. In the situationwhere the experimentor does not know the most useful concentration ofliquids. The initial concentration can be set at a reasonable level, andthe results observed. If unsatisfactory, the concentration can bechanged gradually until the desired or final concentration is achieved,at which point the concentration can be maintained at the desired level.If it is determined that gradient elution, a change from oneconcentration to another, is desirable, but the optimum rate of changeis not known, the rate can be varied simply by programming the valves toopen at a different frequency relative to one another, until the optimumrate of change is found.

FIG. 2 illustrates an improved version of the system shown in FIG. 1.The only change is that pump 14 has been replaced by a liquid supplymeans comprising a holding chamber 25 coupled between valve 17 and pump13. The holding chamber is initially filled with liquid B from source 12by opening valve 24 and allowing the chamber to fill by gravity ofpressure. Valve 24 is then closed, and constant pressure pump 13operates alternately to supply a portion of liquid A is directly tovalve 16 and a portion of liquid A to the holding chamber 25 to forceliquid B, contained in the holding chamber to valve 17. The advantage ofthis system is that a single pump can be used which means that thesystem is less expensive and also that both liquids are supplied totheir respective valves at the same constant pressure or flow rate. Whentwo pumps are used it is different to achieve this latter conditionbecause it is difficult to match the pressures or flow rates developedby different pumps. The holding chamber must be constructed so thatsubstantial mixing of liquid A and liquid B do not occur while liquid Ais being used to force liquid B to valve 17. Also enough of liquid Bmust be contained in the holding chamber to complete the elution run. Along narrow holding chamber, such as a long thin tube fulfills theseconditions.

FIG. 3 illustrates another embodiment of the same system. In thisembodiment, the two valves 16 and 17 have been replaced by a singlethree-way valve 26, and constant pressure pump 13 has been replaced byconstant flow pump 27. Valve 26 is located upstream of holding chamber25 rather than on the downstream side but it could just have easily beenlocated on the downstream side. Operation of the system is the same asthat discussed with respect to FIG. 2.

FIG. 4 illustrates the preferred embodiment of the elution system. Inthis embodiment liquid sources 11 and 12 are provided with sight tubes30 and 31, respectively, so that the level of liquid in each can bemonitored. The holding chamber is in the form of a long thin coil oftubing 34, which is still filled by gravity or pressure through valve24. Air or vacuum can be supplied to liquid source container 12 throughvalves 32 and 33 respectively to assist in filling holding chamber 34.The holding chamber is connected directly to drain 23 through valve 36which is normally closed, and used to both drain the system and to aidin filling the holding chamber. The upstream side (low end) of theholding chamber is connected to pump 13 through valve 35 which is openedwhen the liquids are to be supplied to their respective valves. Theoperation of the system is similar to that discussed with reference toFIG. 2. The holding chamber is filled and valves 24 and 36 are closed.Valve 35 is open and pump 13 is used to both supply liquid A to valve 16and to force the liquid contained in the holding chamber 34 to valve 17.The programmed mixture is forced through the column to a drain 23. Caremust be taken in the operation of the system so that holding chamber 34is not inadvertently emptied of liquid B during protracted operation sothat only liquid A is being supplied to the mixing chamber. To preventthis, some means may be provided to keep track of the amount of liquid,originally contained in the holding chamber, that has been used. Controlmeans 37 and trap 38 have been provided for this purpose. As illustratedcontrol means 37 is a three-way valve. The three-way valve is adapted sothat when valve 17 is open, the three-way valve is in the positionillustrated and a volume of liquid equal to the volume of liquid thathas passed through valve 17 is collected in trap 38, valve 39 beingclosed. When valve 17 is closed, the three-way valve rotates a quarterturn to the left and the liquid passes directly to the drain. The liquidlevel in trap 38 reflects the amount of liquid B removing in the holdingchamber so that it can be replenished before it runs out. When the runis over, trap 38 is drained through valve 39.

It is convenient to have control means 37 activated by the same signalthat activates valve 17. FIG. 5 illustrates a simple way in which thiscan be done. Liquid leaving detector 22 is passed through a flexibletube 53 which is attached to rod 54, which in turn forms part of asolenoid system 55. Two traps are provided. One, 52, leads directly todrain 23, the other, 50, leads to the drain 23 through valve 39 which isusually closed. Trap 50 can be provided with a sight glass 51. Whenvalve 17 is activated by an electrical signal, solenoid 55 is alsoactivated, and flexible tube 53 is positioned over trap 50. When valve17 is closed, solenoid 55 returns flexible tube 53 to its position overdrain 23. This is a convenient way to collect an amount of liquid equalto the volume passing through valve 17, but any convenient way known tothose skilled in the art can be used.

One advantage to all three of the systems described above is that.[.when.]. .Iadd.they alleviate one of the problems which occur in highpressure systems. When .Iaddend.a high pressure elution system is beingused, there is always the problem of high pressure liquid leaks. Leaksfrom the inside to the outside of the valves are not difficult to stop,but leaks through the valve are difficult to stop and require expensivevalving. In the case of the constant pressure system described above,the pressure on both sides of valve 16 and 17 is substantially the same,so there is no pressure differential to cause a leak.

One of the greatest advantages of the present system is its versatility.The initial and final concentration of the eluent can be set with ease,and the rate of change of this concentration can also be set with ease.FIG. 6 illustrates a programming sequence that can be used to change theconcentration in the eluent. This system uses two electronic signals;one signal, signal 60, is a periodic saw tooth wave, the other signal,signal 62, is a gating signal, which in the case illustrated is a linearramp. The programming means, being an electronic signal generator,generates the two signals simultaneously, and is adapted to activate onevalve, when the instantaneous value of the saw tooth wave is greaterthan the instantaneous value of the gating function, and to actuate theother valve when the instantaneous value of the saw tooth wave is lessthan the instantaneous value of the gating function. If valve 16 is thefirst valve, the shaded region of the bar below the graph indicates thetime interval that liquid A is being supplied to the mixing chamber, andthe unshaded regions indicate the time intervals where liquid B is beingsupplied. The relative periods will depend on the types of functionsgenerated. Utilizing the versatility of electronic function generating,almost any programming sequence can be envisioned. The one illustratedis a useful programming sequence. A saw tooth wave 60 combined with anymonotonically increasing gating signal of arbitrary function, such assignal 63 is another useful sequence. A linear gating signal 62,combined with any periodic signal of arbitrary function would also beuseful.

The above discussion has been for the purpose of illustrating theusefulness and operation of our invention. It is claimed that more thantwo liquids can be handled in this manner, and that there are numerousmodifications of the system disclosed that would occur to those skilledin the art. The above discussion is, therefore, not meant to limit thescope of our invention which is set forth in the following claims.

What is claimed is:
 1. A method of producing an eluent containing a timevarying concentration of two liquids, comprising the steps of:a.supplying a first liquid to a first operative portion of normally closedvalving means; b. supplying a second liquid to a second operativeportion of said normally closed valving means; c. alternately activatingsaid first operative portion of said valving means for a first setperiod of time to allow a first set volume of said first liquid to entera mixing region and then activating said second operative portion ofsaid valving means for a second set period of time to allow a second setvolume of said second liquid to enter said mixing region and mix withsaid first set volume of first liquid to form a mixture containing a setconcentration of said first and second liquids.Iadd., said first andsecond set periods of time being such that the volume of said first andsecond liquid entering said mixing region during each period when eitheroperative portion of said valving means is activated is less than thevolume of said mixing region; .Iaddend. d. allowing a portion of saidmixture to leave said mixing region thereby producing an eluent having aconcentration of said first and second liquid, the instantaneousconcentration of which is equal to the instantaneous concentration ofsaid first and second liquids at the downstream end of said mixingregion at the time said portion of said mixture leaves said mixingregion; and e. varying said first and second set periods of time duringwhich said first and second operative portions of said valving means arerespectively activated, whereby the concentrations of said first andsecond liquids in said mixture are gradually changed so that an eluenthaving a time varying concentration of said first and second liquids isproduced.
 2. The method of claim 1 wherein the step of supplying a firstliquid to the first operative portion of the valve means is accomplishedby pumping a first quantity of the first liquid directly to the firstoperative portion of the valve means, and the step of supplying a secondliquid to the second operative portion of the valving means isaccomplished by transferring a quantity of the second liquid to a longnarrow holding chamber and then pumping the second liquid contained inthe holding tube to the second operative portion of the valving means bypumping a second quantity of the first liquid to the upstream side ofthe holding tube.
 3. The method of claim 1 wherein the first and secondliquids are supplied to the first and second operative portions of thevalving means at a constant pressure.